I propose to study the development of the amniote choana, with specific emphasis on identifying putative influences on other key elements of the embryonic oral cavity such as secondary palate formation. The choanae are the internal nares which connect the oral cavity to the nasal passages and are crucial for proper nasal respiration. In mammals, the choanae are covered by the secondary palate and therefore, open directly into the nasopharynx. In most other reptilian amniotes however, the choanae open directly into the oral cavity. Despite their persistence in all vertebrates, very little focus has been placed on this structure. This is especially remarkable since incorrect development of the choana (e.g. retention of the oronasal membrane separating the choana from the nasal passage) in humans, which results in choanal atresia (CA), can lead to infant mortality. Several recent studies have identified genetic pathways which, when perturbed, can cause CA in mouse models. Additionally, our preliminary studies have shown that choanae express key developmental genes which are implicated in various other aspects of cranial development ranging from the initiation of tooth development to the growth and fusion of the secondary palate. I hypothesize that in very young embryos the choanae are in close enough proximity to influence development of the adjacent structures in the roof of the oral cavity. Therefore, I have proposed a series of studies which will serve to characterize the tissue specific changes associated with proper choanal development in amniotes with and without a secondary palate. Subsequently, I seek to identify the likely roles of the molecular pathways which we have observed to be specifically active in the choanae. For these studies, I will use two model organisms, chicken and mouse, as well as a non-model organism, the turtle. These three amniotes represent three different types of secondary palates. Mice have a complete secondary palate, chickens have a natural cleft palate and turtles have primitive palate. These comparative studies will allow us to compare genes expressed in tissues surrounding the choanae and identify those that are correlated with a closed versus open palate. Next, I will culture the embryonic palate containing the choanae and will increase or decrease the levels of different signalling molecules that are enriched in this region. I will also implant microscopic foil barriers to block the choana signals from reaching the palate. The effect of these interventions on the breakdown of the oronasal membrane and the initiation of palatal shelves will be investigated. In chicken, the longer term effects on palatine bones will be studied by adding the signals or their inhibitors to the embryos while in the egg. These will be the first studies on the embryonic development of the choanae. The presence of a new signaling center in the face will be investigated, and novel molecular causes for facial abnormalities such as cleft palate will be discovered.